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DNA-Templated Native Chemical Ligation of Functionalized Peptide Nucleic Acids: A Versatile Tool for Single Base-Specific Detection of Nucleic Acids

  • Alexander Roloff
  • Simon Ficht
  • Christian Dose
  • Oliver Seitz
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1050)

Abstract

Single base-specific detection of DNA/RNA sequences is of importance in the diagnosis of disease-associated genetic disorders or early stage cancer. This chapter introduces DNA-templated native chemical PNA ligation as a potentially useful tool for the sequence specific detection of nucleic acids. The template-induced alignment of PNA-thioesters and 1,2-aminothiol-PNAs in close proximity leads to an increase in their effective molarities. This facilitates PNA ligation to proceed at concentrations where no reaction would be possible in absence of the template. Moreover, hybridization of the rather short PNA conjugates with non-complementary DNA/RNA is disfavored, which prevents PNA ligation to occur on single base-mismatched templates. Different readout strategies of the ligation reaction such as HPLC, MALDI-TOF-MS and fluorecence monitoring are discussed, and examples for the detection of a point mutation within single stranded and PCR-amplified double stranded DNA are provided.

Key words

DNA template Native chemical ligation PNA, Single base specificity Templated chemistry 

References

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Alexander Roloff
    • 1
  • Simon Ficht
    • 1
  • Christian Dose
    • 1
  • Oliver Seitz
    • 2
  1. 1.Department of Organic and Bioorganic ChemistryHumboldt-Universität zu BerlinBerlinGermany
  2. 2.Department of ChemistryHumboldt-Universität zu BerlinBerlinGermany

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